Field of the Invention
The present invention relates to the development of genetically engineered microorganisms that can produce wax esters in a controllable and economic fashion. More specifically the invention relates to the production of liquid wax esters that can be used for biofuel, lubricants, cosmetics, linoleum, printing inks as well as products related thereto, and for the production of solid wax esters used for candles and polishes as well as products related thereto.
Description of the Related Art
Fossil fuels, such as coal, oil, and natural gas, have been powering modern society for more than one century. However, fresh discoveries of deposits are on the wane and demands are increasing. The world's demand of fossil fuels will soon outweigh the current supply. An innovative approach offering some solution comes from the biotechnology industries. Efforts have made biodiesel as one of the most thoroughly developed and promising alternative fuels on the market. It works well in conventional diesel engines, with less hazardous emissions, and is consumed at greater than 3.5 billion gallons per year.
Biodiesel is generally composed of fatty acid methyl esters (FAMEs) or fatty acid ethyl esters (FAEE), and is mostly derived from vegetable oil or animal fat by chemically transesterification with methanol or ethanol. Despite the fact that ethanol-yielded FAEEs have better performances, for cost reasons methanol is the reagent most frequently used for triglyceride transesterification. The current process has several drawbacks, including energy intensiveness, consuming edible feedstocks, difficulty of removal of the catalyst from the product and treatment of toxic waste-water, as well as geographical and seasonal restrictions.
To overcome the problems related to the use of catalysts people have been exploring new alternatives such as enzymatic conversion using lipases (EC 3.1.1.3, triacylglycerol hydrolases). Lipases can break down neutral lipids such as triglycerides and perform a transesterification reaction in a solvent system (i.e. tert-butanol). Enzymatic production of biodiesel can be carried out at moderate reaction conditions and at a lower alcohol to oil ratio. The main drawbacks with this kind of enzymatic catalysis are the strong inactivation effect caused by alcohols (i.e. methanol) and the high enzyme costs.
Both chemical and enzymatic transesterification require the use of toxic, petrochemically-derived alcohols and expensive feedstocks. Thus, transesterification-based biodiesel becomes unsustainable when fossil fuel derived products are used. As a result, the current feedstocks for biodiesel are mainly derived from plant oils like rapeseed oil.
However such plant oils are inherently limited by supply of water and land, and subsequently, they cannot produce enough biofuel without threatening food supplies and/or native biodiversity. Algae are a promising choice as an alternative feedstock. Nevertheless, there are problems with surface usage and oil extraction from algae based production. Everyone agrees that fuels derived from biomass are one of the best alternatives to fossil fuels. Thus, genetically manipulation of microorganisms to produce fatty acid esters, will substantially contribute to produce environmentally friendlier, sustainable, and cost-effective biodiesels.
In this regard, it was previously shown that an engineered E. coli strain expressing the wax synthase (WS) from Acinetobacter baylyi ADP1 and ethanol-production genes from Z. mobilis, could produce fatty acid ethyl esters by esterifying exogenously added fatty acids (Kalscheuer, Stolting et al. 2006). The research is an excellent demonstration of feasibility for microbial production of fatty acid esters. Recently, researchers from the Keasling group and the company LS9 Inc. (South San Francisco, USA) developed this idea further by constructing an engineered E. coli that can produce fatty-acid-derived fuels and chemicals from simple sugars and plant-derived biomass, without the need for fatty acid feeding (Steen, Kang et al. 2010). Production of fatty acid derivatives as biofuels has also been reported in recent patent applications WO2009/009391, WO2007136762 and WO2008119082, all owned by LS9 Inc. Briefly, the metabolically engineered E. coli strain was manipulated to be able to produce fatty (acid) esters and derivatives thereof (short and long chain alcohols, hydrocarbons, fatty alcohols, waxes, etc.) through the introduction of several genes encoding for enzymes such as thioesterase, wax synthase, alcohol acyltransferase, alcohol dehydrogenase, and different kinds of fatty alcohol forming acyl-CoA reductases. In U.S. patent publication 2010/0071259, inventors from the same company teach that by adding a mixture of at least two different alcohols to a medium containing the engineered E. coli strain that produces fatty esters, at least two different fatty esters could be produced.
The afore-mentioned biodiesel producing methods are all based on the use of the bacterium E. coli. However, E. coli is unable to naturally overproduce the two substrates of biodiesel, fatty acids and alcohol (i.e. ethanol), and this organism is not suitable for large-scale production that often involves harsh environmental conditions. Furthermore, E. coli is sensitive to phage contamination often resulting in substantial economic losses. The patents of the prior art successfully teach several strategies to enhance fatty acids biosynthesis in E. coli. Nevertheless, and apart from the drawbacks associated with the use of this host, it should be noted that strategies working in E. coli might not be appropriate when applied in other microorganisms.
A far better choice of microbial cell factory for industrial production of biodiesel would be the yeast Saccharomyces cerevisiae. This yeast is already widely used in industry, including for large-scale bioethanol production, but also for a range of specialty chemicals. The development of S. cerevisiae as a cell factory for biodiesel production would represent a major contribution as this could represent a plug and play solution where current infrastructures used for production of bioethanol could be used for production of far more valuable biodiesels. In contrast to the insufficient ethanol productivity of E. coli, S. cerevisiae is already a good ethanol producer.
In fact, production of FAEEs and fatty acid isoamyl esters (FAIEs) has been achieved in recombinant S. cerevisiae with oleic acid addition by expressing the A. baylyi bifunctional WS/DGAT enzyme (Kalscheuer, Luftmann et al. 2004). A recent patent application, namely US patent application 2009/0117629 by Schmidt-dannert and Holtzapple, also describes a method for the production of esters, including isoprenoid wax esters and fatty acid alkyl esters, such as FAME and FAEE, by heterologous expression of Marinobacter hydrocarbonoclasticus wax synthase (WS2) in S. cerevisiae. The invention is however, limited to the use of specific isolated polynucleotides from Marinobacter hydrocarbonoclasticus, and its application in e.g. producing biodiesel). Moreover this method requires exogenous supply of fatty acids as the endogeneous production of fatty acids by yeast is too low to ensure economically viable production of FAEEs.
A modified strain carrying the genes encoding the wax synthase from Marinobacter hydrocarbonoclasticus could be considered a potential host for biodiesel production in yeasts. Nonetheless, while this product is very suitable for the particular purpose it addresses, it is not the ideal option when the synthesis of other esters is desired. The knowledge of the preferred substrates for each wax synthase allows the use of yeast cells in applications other than biodiesel production. Moreover there is still a need for methods and products allowing large-scale production of fatty acid esters.
Thus it is an object of the present invention to provide an improved fungal cell factory, such as a yeast cell factory that can be used for fermentation based production of FAEEs, that is not dependent on the addition of exogenous fatty acids to the yeast culture and that possess an increased flux towards fatty acid biosynthesis and where high level production of FAEEs is obtained.